RESUMEN
AIM: The present study was thus undertaken to analyze the genetic diversity of Kilakarsal and Vembur sheep breeds using single-nucleotide polymorphism (SNP) markers within Toll-like receptor (TLR) 3, 5, 6, 9, and 10 genes. MATERIALS AND METHODS: Competitive allele-specific polymerase chain reaction (PCR)-based end-point genotyping was performed using real-time PCR to type the SNPs. Allele discrimination module implemented in real-time PCR was utilized to call the genotypes based on fluorescence intensity recorded for each of the two alleles. Basic diversity indices, namely, gene frequencies, observed heterozygosity, expected heterozygosity, and inbreeding coefficient (FIS), and testing for Hardy-Weinberg equilibrium (HWE) were estimated using package for elementary analysis of SNP data software program. RESULTS: Of the 25 SNPs, 22 were found to be polymorphic, whereas two SNPs, namely, TLR3_1081_AC and TLR9_2036_CT, were monomorphic in both Kilakarsal and Vembur sheep populations. The SNP TLR10_1180_AG was monomorphic in Kilakarsal but polymorphic in Vembur sheep. The observed heterozygosities were estimated as 0.289 and 0.309 in Kilakarsal and Vembur sheep, respectively, whereas the expected heterozygosity values were 0.305 and 0.309 in the two breeds, respectively. The overall mean FIS was 0.107 ranging from -0.005 to 0.241 in Kilakarsal sheep and -0.047 ranging from -0.005 to 0.255 in Vembur sheep. In Kilakarsal sheep, the test for HWE revealed TLR9_1308_GC SNP locus with significant deviation (p<0.05) due to heterozygosity deficit. In Vembur sheep, TLR10_82_CT and TLR10_292_CG loci showed significant deviation (p<0.05) due to heterozygosity excess. Other SNP loci did not deviate from HWE (p>0.05) revealing that the population was in HWE proportions. CONCLUSIONS: The SNP markers within five TLR genes (TLR3, TLR5, TLR6, TLR9, and TLR10) utilized for genotyping in this study were highly polymorphic in Kilakarsal and Vembur breeds of sheep. This study on the genetic diversity analysis of the Kilakarsal and Vembur sheep breeds revealed considerable genetic variation within the breeds and it can be utilized to improve desirable traits.
RESUMEN
BRAF mutations exist in numerous types of cancer, including melanomas, colorectal cancers and lung cancers. The V600E-specific inhibitor vemurafenib has marked clinical activity in patients with BRAF V600E-mutated melanoma. However, there are many cases of resistance to vemurafenib. This may be due to the reported intra-tumor heterogeneity of the BRAF V600E mutation in primary melanomas. BRAF mutations are found in 1-5% of non-small cell carcinomas (NSCLCs), almost exclusively in adenocarcinoma. A few cases have been reported in which vemurafenib was effective against BRAF V600E-mutated lung cancers. In a previous study, five lung adenocarcinomas with BRAF V600E mutation were detected by direct sequencing. The present study analyzed these tumors for the percentage of mutation (%mutation) by competitive allele-specific polymerase chain reaction (CAST-PCR) assay. In addition, sections of all components of the adenocarcinomas were obtained by laser microdissection and analyzed. The %mutations of BRAF V600E within the macrodissected tumors (cases 1-5) were: Case 1, 10.0%; case 2, 8.0%; case 3, 8.9%; case 4, 21.5%; and case 5, 14.9%. In four cases (cases 2-5), the %mutations of each adenocarcinoma component were as follows: Case 2, lepidic growth 6.5-24.5%, papillary 1.3-11.2% and acinar 9.8%; case 3, solid 2.5-69.9%, acinar 12.4-27.1% and papillary 3.7-17.4%; case 4, acinar 10.0-45.0% and papillary 44.0%; and case 5, papillary 3.7-93.4%. Sensitive BRAF mutation detection methods were used and evidence for heterogeneity of the BRAF V600E mutation in these lung adenocarcinoma cases was observed. Targeted therapy with a BRAF V600E inhibitor such as vemurafenib may have potential in the treatment of lung cancer with this mutation; however, it is necessary to consider how the treatment effect of and drug resistance to BRAF V600E inhibitors are affected by the presence of heterogeneity in future studies.